3.4 High performance liquid chromatography HPLC

3.4 High performance liquid chromatography HPLC

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Unformatted text preview: glass culture tube using 5 mL of 5 % ammonium hydroxide in methanol. The eluate was evaporated to dryness at 55 °C under flowing nitrogen at 15 psi for 20 minutes. The dried extract The dried was reconstituted in 1.0 mL of 95:5 acetonitrile:ammonium formate (20 mM), vortex mixed for 10 seconds, and filtered through a 0.2 µm nylon syringe filter into a glass LC vial. vial. 1/11/2012 CHEM2202 WTCHAN 33 Determination of Melamine Residues in Catfish Tissue by Triple Quadrupole LC-MSMS with HILIC Chromatography – US FDA (http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/DrugChemicalResiduesMethodology/ucm071796.htm) (http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/DrugChemicalResiduesMethodology/ucm071796.htm) LC‐MS‐MS Analysis The LC‐MS‐MS electrospray conditions were optimized by tuning with a 10 ng/µL solution of MEL (melamine) flowing into the mass spectrometer with a syringe pump at 10 µL/min while 350 µL/min of 75:25 acetontrile:ammonium formate buffer (20 mM) was added via a T‐union. The combined stream was introduced into the electrospray interface. 1/11/2012 CHEM2202 WTCHAN 34 http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/DrugChemicalResiduesMethodology/ucm071796.htm http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/DrugChemicalResiduesMethodology/ucm071796.htm 1/11/2012 CHEM2202 WTCHAN 35 1/11/2012 CHEM2202 WTCHAN 36 1/11/2012 CHEM2202 WTCHAN 37 In ion chromatography (ionexchange chromatography), the stationary phase is an ion-exchange resin The exchange equilibrium occurs between the liquid mobile phase and the solid stationary phase: − − xRSO3 H + solid + M x + solution ↔ (RSO3 ) x M x + solid + xH + solution Ions of like charge are separated by elution from a column packed with a finely divided resin. 1/11/2012 CHEM2202 WTCHAN 39 Ion-exchange resins are high molecular weight polymeric materials containing many ionic functional groups per molecule Cation exchange resins 1.. 1 2. Strong-acid type with sulfonic acid groups (RSO3H) Weak-acid type with carboxylic acid groups (RCOOH) Anion exchange resins 1.. 1 2. 1/11/2012 Strong-base exchangers are quaternary amines Weak-base types contain secondary or tertiary amines CHEM2202 WTCHAN 40 In ion chromatography, the concentration of H+ ions in both mobile phase and stationary phase can be considered constant. The concentration of H+ ions is much larger than the metal ions in both phases and [H+] in the phases do not change significantly with the introduction of Mx+ K' = x [M x + ]res [H + ]aq x [M x + ]aq [H + ]res x [M x + ]res [H + ]res = K' + x = K x+ [M ]aq [H ]aq 1/11/2012 CHEM2202 WTCHAN 41 Retention of an ion depends strongly on the charge on the ion and the size of the hydrated ion Polyvalent ions are much more strongly retained than singly Polyvalent charged species Within a given charge group, K (partition coefficient) reduces Within as the size of the hydrated ions increases size increases For univalent ions, the retention order is: retention Ag+ > Cs+ > Rb+ > K+ > NH4+ > Na+ > H+ > Li+ For divalent cations, the order is: Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ > UO22+ 1/11/2012 CHEM2202 WTCHAN 42 1/11/2012 CHEM2202 WTCHAN 43 1/11/2012 CHEM2202 WTCHAN 44 In size-exclusion chromatography (gel chromatography), fractionation is based upon molecular size. It is a powerful technique for the separation of species of high-molecular weight. 1/11/2012 CHEM2202 WTCHAN 45 The packing is small (10 μm) silica or polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse. While in the pores, molecules are effectively trapped and removed from the flow of the mobile phase. 1/11/2012 CHEM2202 WTCHAN 46 1/11/2012 CHEM2202 WTCHAN 47 Two-dimensional TLC 1/11/2012 CHEM2202 WTCHAN 49...
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This note was uploaded on 08/17/2013 for the course CHEM 2202 taught by Professor Wt during the Fall '11 term at HKU.

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